Abstract:

A gene expressing cassette codes lactate dehydrogenase that is needed for
prevention of deterioration in lactic acid yield and lactic acid
production rate in continuous culture with simultaneous filtration of a
yeast strain having a lactic acid-producing ability, which achieves high
optical purity, high lactic acid yield and high lactic acid production
rate simultaneously, a yeast strain having the cassette and a method of
producing lactic acid by culturing the yeast strain. The lactate
dehydrogenase-expressing cassette is a lactate dehydrogenase-expressing
cassette, comprising a gene coding lactate dehydrogenase connected to a
site downstream of a promoter, the promoter being a promoter of a gene
showing a gene expression amount larger by 5 times or more than the
average relative expression amount of all genes after 50 hours from start
of culture in continuous culture with simultaneous filtration of a yeast
strain having a lactic acid-producing ability.

Claims:

1. A lactate dehydrogenase-expressing cassette, comprising a gene coding
lactate dehydrogenase connected to a site downstream of a promoter,
wherein the promoter is a promoter of a gene showing a gene expression
amount larger by 5 times or more than the average relative expression
amount of all genes after 50 hours from start of culture in continuous
culture with simultaneous filtration of a yeast strain having a lactic
acid-producing ability.

3. The lactate dehydrogenase-expressing cassette according to claim 1,
wherein the promoter is a promoter having a nucleotide sequence selected
from the following sequences (a) to (c):(a) a promoter having the
nucleotide sequence shown by any one of SEQ ID Nos. 1 to 3;(b) a promoter
having a nucleotide sequence that hybridizes with the nucleotide sequence
shown by any one of SEQ ID Nos. 1 to 3 or a nucleotide sequence having
part of it under stringent condition; and(c) a promoter having a
nucleotide sequence obtained by deletion, substitution and/or insertion
of one or more bases from the nucleotide sequence shown by any one of SEQ
ID Nos. 1 to 3.

4. A lactate dehydrogenase-expressing cassette, comprising a promoter
selected from the following group (a) and a gene coding lactate
dehydrogenase selected from group (b):(a)(1) a promoter having the
nucleotide sequence shown by any one of SEQ ID Nos. 1 to 3;(2) a promoter
having a nucleotide sequence that hybridizes with the nucleotide sequence
shown by any one of SEQ ID Nos. 1 to 3 or a nucleotide sequence
containing part of it under stringent condition; and(3) a promoter having
a nucleotide sequence obtained by deletion, substitution and/or insertion
of one or more bases from the nucleotide sequence shown by any one of SEQ
ID Nos. 1 to 3,(b)(1) a gene coding lactate dehydrogenase having the
nucleotide sequence shown by any one of SEQ ID Nos. 4 to 6;(2) a gene
coding lactate dehydrogenase having a nucleotide sequence that hybridize
with the nucleotide sequence shown by any one of SEQ ID Nos. 4 to 6 or a
nucleotide sequence containing part of it under stringent condition;
and(3) a gene coding lactate dehydrogenase having a nucleotide sequence
obtained by deletion, substitution and/or addition of one or more bases
from the nucleotide sequence shown by any one of SEQ ID Nos. 4 to 6.

5. A transformant yeast strain, comprising at least one lactate
dehydrogenase-expressing cassette according to claim 1 on a chromosome.

6. The transformant yeast strain according to claim 5, wherein at least
one gene thereof selected from suppression-of-exponential-defect 1 gene
(SED1 gene), cell-wall-associated protein 2 gene (CWP2 gene) and enolase
1 gene (ENO1 gene) is substituted with the lactate
dehydrogenase-expressing cassette that comprises a gene coding lactate
dehydrogenase connected to a site downstream of a promoter, wherein the
promoter is a promoter of a gene showing a gene expression amount larger
by 5 times or more than the average relative expression amount of all
genes after 50 hours from start of culture in continuous culture with
simultaneous filtration of a yeast strain having a lactic acid-producing
ability.

9. A method of producing lactic acid, comprising a culture step of
culturing the transformant yeast strain according to claim 5.

10. The method of producing lactic acid according to claim 9, wherein the
culture step is a continuous fermentation comprising filtering a culture
solution through a separation membrane, recovering lactic acid from
resulting filtrate, holding or feeding back unfiltered solution to the
culture solution, and adding medium to the culture solution.

Description:

RELATED APPLICATIONS

[0001]This is a §371 of International Application No.
PCT/JP2008/072129, with an international filing date of Dec. 5, 2008 (WO
2009/072593 A1, published Jun. 11, 2009), which is based on Japanese
Patent Application No. 2007-317566, filed Dec. 7, 2007, the subject
matter of which is incorporated by reference.

TECHNICAL FIELD

[0002]This disclosure relates to a lactate dehydrogenase-expressing
cassette, a transformant yeast strain containing the cassette and a
method of producing lactic acid comprising culturing the yeast strain,
more specifically, a lactate dehydrogenase-expressing cassette higher in
lactic acid production efficiency, a transformant yeast strain containing
the cassette and a method of producing lactic acid comprising culturing
the yeast strain.

BACKGROUND

[0003]Recently, polymers that are prepared by using biomasses such as
plants as raw material are attracting attention in a trend toward
establishment of a resource-circulating society. In particular,
polylactic acid (hereinafter, referred to as "PLA") was shown to have
favorable properties as a polymer from biomass raw material.

[0004]Lactic acid, the raw material for PLA, is produced by fermentation
of microbes, generally called lactic bacteria, represented by those of
Lactobacillus species and Lactococcus species. Production of lactic acid
by using lactic bacteria is superior in lactic acid yield from sugar and
lactic acid production rate. However, the lactic acid obtained is a
mixture of L- and D-lactic acids. Thus, there is a problem in optical
purity. High optical purity is demanded for the lactic acid for use in
production of PLA.

[0005]There were several attempts to produce high-optical purity lactic
acid. For example, production of L- and D-lactic acids by using a
transformant yeast strain was studied (for example, JP 2001-516584 A, JP
2003-093060 A, JP 2005-137306 A and N. Ishida et al., J. Biosci. Bioeng.,
101(2), pp. 172-177, 2006). Yeasts do not have ability to produce lactic
acid inherently. It was reported in these literatures that high-optical
purity lactic acid could be obtained by introducing a gene coding lactate
dehydrogenase, which converts pyruvic acid into lactic acid, into yeast
by genetic recombination technology. On the other hand, the lactic acid
yield and the lactic acid production rate during lactic acid production
by yeast are lower, compared to those by lactic bacteria. It is thus
needed to improve both lactic acid yield and lactic acid production rate
for production of lactic acid by using a yeast at low cost.

[0006]For improvement of the lactic acid yield and the lactic acid
production rate at the same time, a method of culturing a yeast strain
having a lactic acid-producing ability while the fermentation solution is
filtered through a separation membrane was developed (see, for example,
WO 2007/97260). However, even if the method was used, it caused a problem
that both the lactic acid yield and the lactic acid production rate
declined during fermentation.

[0007]It could therefore be helpful to provide a gene expressing cassette
coding lactate dehydrogenase that is needed for prevention of
deterioration in lactic acid yield and lactic acid production rate in
continuous culture with simultaneous filtration of a yeast strain having
a lactic acid-producing ability, which achieves high optical purity, high
lactic acid yield and high lactic acid production rate simultaneously, a
yeast strain having the cassette and a method of producing lactic acid by
culturing the yeast strain.

SUMMARY

[0008]We found that it was possible to carry out continuous culture
consistently for an extended period of time without deterioration in
lactic acid yield and lactic acid production rate, by culturing a yeast
strain having a lactate dehydrogenase-expressing cassette containing a
promoter of a gene showing a gene expression amount larger by 5 times or
more than the average relative expression amount of all genes after 50
hours from start of culture while filtering the yeast through a
separation membrane, in continuous culture with simultaneous filtration
by separation membrane of a yeast strain having a lactic acid-producing
ability.

[0009]We thus provide a lactate dehydrogenase-expressing cassette,
comprising a gene coding lactate dehydrogenase connected to a site
downstream of a promoter, the promoter being a promoter of a gene showing
a gene expression amount larger by 5 times or more than the average
relative expression amount of all genes after 50 hours from start of
culture in continuous culture with simultaneous filtration of a yeast
strain having a lactic acid-producing ability. Preferably, the promoter
is the promoter of suppression-of-exponential-defect 1 gene (SED1 gene),
cell-wall-associated protein 2 gene (CWP2 gene) or enolase 1 gene (ENO1
gene), more preferably, a promoter having a nucleotide sequence selected
from the following sequences (a) to (c): [0010](a) a promoter having
the nucleotide sequence shown by any one of SEQ ID Nos. 1 to 3; [0011](b)
a promoter having a nucleotide sequence that hybridizes with the
nucleotide sequence shown by any one of SEQ ID Nos. 1 to 3 or a
nucleotide sequence having part of it under stringent condition; and
[0012](c) a promoter having a nucleotide sequence obtained by deletion,
substitution and/or insertion of one or more bases from the nucleotide
sequence shown by any one of SEQ ID Nos. 1 to 3.

[0013]We also provide a lactate dehydrogenase-expressing cassette
containing a promoter selected from the following group (a) and a gene
coding lactate dehydrogenase selected from the following group (b):

(a) [0014](1) a promoter having the nucleotide sequence shown by any one
of SEQ ID Nos. 1 to 3; [0015](2) a promoter having a nucleotide sequence
that hybridizes with the nucleotide sequence shown by any one of SEQ ID
Nos. 1 to 3 or a nucleotide sequence containing part of it under
stringent condition; and [0016](3) a promoter having a nucleotide
sequence obtained by deletion, substitution and/or insertion of one or
more bases from the nucleotide sequence shown by any one of SEQ ID Nos. 1
to 3, and(b) [0017](1) a gene coding lactate dehydrogenase having the
nucleotide sequence shown by any one of SEQ ID Nos. 4 to 6; [0018](2) a
gene coding lactate dehydrogenase having a nucleotide sequence that
hybridize with the nucleotide sequence shown by any one of SEQ ID Nos. 4
to 6 or a nucleotide sequence containing part of it under stringent
condition; and [0019](3) a gene coding lactate dehydrogenase having a
nucleotide sequence obtained by deletion, substitution and/or addition of
one or more bases from the nucleotide sequence shown by any one of SEQ ID
Nos. 4 to 6.

[0020]We further provide a transformant yeast strain, comprising at least
one lactate dehydrogenase-expressing cassette described above on
chromosome. Preferably, it is a yeast in which at least one gene selected
from suppression-of-exponential-defect 1 gene (SED 1 gene),
cell-wall-associated protein 2 gene (CWP2 gene) and enolase 1 gene (ENO1
gene) is substituted with the lactate dehydrogenase-expressing cassette.

[0022]We still further provide a method of producing lactic acid
comprising a culture step of culturing the transformant yeast strain.
Preferably, the culture step is continuous fermentation of filtering the
culture solution through a separation membrane, recovering lactic acid
from the filtrate, holding or feeding back the unfiltered solution to the
culture solution, and adding the medium to the culture solution.

[0023]A yeast strain having a lactate dehydrogenase-expressing cassette
containing the promoter of a gene showing a gene expression amount larger
by 5 times or more than the average relative expression amount of all
genes after 50 hours from start of culture is cultured, while the culture
solution is filtered through a separation membrane, in continuous culture
with simultaneous filtration of the yeast strain having a lactic
acid-producing ability. It is possible as a result to produce
high-optical purity lactic acid at high yield and high production rate
consistently for an extended period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a schematic side view explaining a membrane-separation
continuous fermentation apparatus.

[0025]FIG. 2 is a schematic side view explaining another continuous
fermentation apparatus for use in another membrane-separation.

[0051]The lactate dehydrogenase-expressing cassette is a lactate
dehydrogenase (herein-after, referred to as "LDH")-expressing cassette,
in which a gene coding lactate dehydrogenase (ldh gene) is connected to a
site downstream of the promoter thereof, wherein, the promoter is a gene
promoter having a gene expression amount larger by 5 times or more than
the average relative expression amount of all genes after 50 hours from
start of culture, in continuous culture with simultaneous filtration by
separation membrane of a yeast strain having a lactic acid-producing
ability.

[0054]The ldh genes include mutant genes for example caused by genetic
polymorphism and mutagenesis. The genetic polymorphism, as used herein,
is partial change of the nucleotide sequence of a gene caused by natural
mutation of the gene. Alternatively, the mutagenesis is artificial
mutation of a gene, and performed, for example, by a method of using a
site-specific mutation kit (Mutan-K (manufactured by Takara Bio Inc.)) or
a method of using a random mutation kit (BD Diversify PCR Random
Mutagenesis (manufactured by CLONTECH)).

[0055]The method of cloning the ldh gene is not particularly limited, and
any known method may be used. Examples thereof include a method of
amplifying and obtaining a desired gene region by PCR (Polymerase Chain
Reaction), based on known gene information, a method of cloning it by
using homology and enzyme activity in genome libraries and cDNA libraries
as indicators, and the like. Alternatively, it may be prepared by
chemical synthesis or genetic engineering, based on known protein
information.

[0056]The lactate dehydrogenase (LDH) expression cassette is not
particularly limited, if it is a nucleotide sequence that can express LDH
from the ldh gene, via mRNA, in the cells containing the LDH-expressing
cassette. Preferably, it is a nucleotide sequence having a promoter, a
ldh gene and a terminator as they are aligned continuously. The
terminator, as used herein, means a sequence that terminates mRNA
transcription of the gene and is normally a 3 terminal-sided downstream
sequence of the gene present on chromosome.

[0057]The "yeast strain having a lactic acid-producing ability" is a yeast
that can produce lactic acid by consumption of sugars such as glucose,
sucrose, fructose and so on, preferably, a yeast containing an ldh gene
introduced by genetic recombination.

[0058]Hereinafter, the method of introducing a ldh gene into yeast will be
described. The ldh gene is introduced into yeast, for example, by a
method of transforming a yeast with a ldh gene expression vector
containing a ldh gene recombined into the expression vector, a method of
inserting a ldh gene at a desired position of chromosome by homologous
recombination, or a method of inserting a ldh gene into chromosome at
random by heterologous recombination.

[0059]Expression vectors commonly used in yeast can be used as the
expression vectors for recombination of the ldh gene. The expression
vector commonly used in yeast has a sequence needed for autonomous
replication in yeast cells, a sequence needed for autonomous replication
in Escherichia coli cells, a yeast selectable marker and an Escherichia
coli selectable marker, and preferably has additionally so-called
regulatory sequences regulating the expression of the recombinant ldh
gene such as of operator, promoter, terminator and enhancer.

[0060]The sequence needed for autonomous replication in yeast cells is,
for example, the sequence in combination of a yeast autonomous
replicating sequence (ARS1) and a centromere sequence or the sequence of
the replication origin of a yeast 2 μm plasmid. The sequence needed
for autonomous replication in Escherichia coli is, for example, the
sequence of the ColE1 replication origin of Escherichia coli.
Alternatively, examples of the yeast selectable markers include
auxotrophic complementary gene such as URA3 and TRP 1 and drug-resistance
genes such as G418 resistance gene and neomycin resistance gene. Examples
of the Escherichia coli selectable marker include antibiotic resistance
genes such as ampicillin resistance gene and kanamycin resistance gene.
The regulatory sequence is not particularly limited, if it is a sequence
that can express the ldh gene, and examples thereof include promoter
sequences such as of acid phosphatase gene (PHO5),
glyceraldehyde-3-phosphate dehydrogenase genes (TDH1, 2 and 3), alcohol
dehydrogenase genes (ADH1, 2, 3, 4, 5, 6 and 7), galactose
metabolism-related genes (GAL1, 7 and 10), cytochrome c gene (CYC1),
triosephosphate isomerase gene (TPI1), phosphoglycerate kinase gene
(PGK1), phosphofructose kinase gene (PFK1), pyruvate decarboxylase genes
(PDC1, 5 and 6) and terminator sequences such as of TDH3 gene. However,
the expression vector is not limited thereto.

[0061]It is possible to obtain a vector capable of expressing a ldh gene
by introducing the ldh gene at a site downstream of the promoter of the
expression vector. It is possible to introduce the ldh gene into yeast by
transforming the yeast with the ldh gene expression vector obtained by
the method described below.

[0062]It is also possible to introduce a ldh gene into yeast by inserting
the ldh gene into chromosome. The method of inserting the ldh gene into
chromosome is not particularly limited, but the ldh gene can be inserted,
for example, by a method of transforming yeast with a ldh gene-containing
DNA by the method described below and inserting the ldh gene at a random
position in chromosome by heterologous recombination or by a method of
inserting a ldh gene-containing DNA at a desirable position by homologous
recombination. It is preferably the method by homologous recombination.

[0063]The method of inserting a ldh gene-containing DNA at a desired
position in chromosome by homologous recombination is, for example, a
method of performing PCR by using a primer designed to add a homologous
region at desired positions upstream and downstream of the ldh
gene-containing DNA and transforming a yeast with the PCR fragments
obtained by the method described below, but is not limited thereto. In
addition, the PCR fragment preferably contains a yeast selectable marker
for easy selection of the transformant.

[0064]The PCR fragment for use is prepared, for example, in the steps of 1
to 3, as shown below in (1) to (3). Here, a method of introducing a ldh
gene at a position downstream of the promoter of pyruvate decarboxylase 1
gene (PDC1 gene) will be described as an example. [0065](1) Step 1: A
fragment containing a ldh gene and a terminator is amplified by PCR, by
using a template of a plasmid having a terminator connected downstream of
a ldh gene as template and a set of primers 1 and 2. Here, the primer 1
is designed to add a homologous sequence of 40 by or more at a position
upstream of the PDC 1 gene, while the primer 2 is designed, based on the
plasmid-derived sequence downstream of the terminator. [0066](2) Step 2:
A fragment containing a yeast selectable marker is amplified by PCR, by
using a yeast selectable marker-containing plasmid, such as pRS424 or
pRS426, as template and a set of primers 3 and 4. Here, the primer 3 is
designed to add a sequence of 30 by or more that is homologous to the
sequence downstream of the terminator in the PCR fragment of Step 1,
while the primer 4 is designed to add a sequence of 40 by or more that is
homologous to the downstream side of the PDC1 gene. [0067](3) Step 3: A
PCR fragment containing a ldh gene, a terminator and a yeast selectable
marker, to which the sequences corresponding to the upstream and
downstream sides of the PDC 1 gene are added at both terminals, is
obtained by preforming PCR by using a mixture of the PCR fragments
obtained in Steps 1 and 2 as template and a set of primers 1 and 4.

[0068]Preferably for introduction of the LDH-expressing cassette into
chromosome, a plasmid carrying a any promoter, a ldh gene and a
terminator is used as the PCR template plasmid used in the Step 1, and
the primer 1 is designed to add a homologous sequence of 40 by or more at
a desired introduction position for amplification of the promoter, the
ldh gene and the terminator.

[0069]A method, for example, of transformation, transduction,
transfection, cotransfection or electroporation may be used for
introduction of the ldh gene expression vector or the PCR fragment thus
obtained into yeast. Typical examples thereof include a method of using
lithium acetate, a protoplast method, and the like.

[0070]The transformant obtained may be cultured by any known method, for
example, by the method described in "M. D. Rose et al., "Methods in Yeast
Genetics", Cold Spring Harbor Laboratory Press (1990)." The yeast
carrying the ldh gene expression vector or the PCR fragment introduced
may be selected, based of the yeast selectable marker contained in the
expression vector or the PCR fragment, when the yeast is cultured in a
nutrient-free medium or a drug-added medium.

[0071]"Continuous culture with simultaneous filtration" means continuous
culture in which the culture solution is filtered through a separation
membrane, the product is recovered from the filtrate, the unfiltered
solution is held in or sent back to the culture solution, and raw
fermentation materials are added to the culture solution. The separation
membrane for use is preferably a porous membrane that is resistant to
clogging by the yeast used in culture and thus gives favorable filtration
performance consistently for an extended period of time. The material for
the separation membrane is a ceramic material or an organic polymer,
preferably an organic polymer.

[0072]Hereinafter, the "gene expressed in a gene expression amount larger
by 5 times or more than the average relative expression amount of all
genes" will be described. The gene expression amount means the amount of
the messenger RNA (mRNA) transcripted from a gene, and the average
relative expression amount of all genes is the average expression amount
of preferably all genes registered in Saccharomyces Genome Database
(www.yeastgenome.org), but one or more genes may be missing. Thus, the
"gene expressed in a gene expression amount larger by 5 times or more
than the average relative expression amount of all genes" means a gene
that is transcripted to its mRNA in an amount larger by 5 times or more
than the average amount of the mRNAs of all genes. Because this
disclosure is characterized by use of a promoter of a gene having a
larger gene expression amount, the gene is preferably a gene that is
expressed in a gene expression amount larger by 7 times or more, more
preferably 10 times or more, than the average relative expression amount
of all genes.

[0073]Examples of the method of determining the average relative
expression amount of all genes include Northern blotting method, qPCR
(quantitative PCR) method, real-time PCR method, DNA microarray method
and the like. Although the former three methods are methods normally for
measuring the expression amounts of individual genes, the DNA microarray
method is a method of measuring the expression amount of all genes
present on an array by means of hybridization of a probe immobilized on
the array with previously fluorescent-labeled mRNAs and measurement of
the fluorescence intensity with a special scanner, and for that reason,
use of the DNA microarray method is preferable. During measurement of the
fluorescence intensity with a scanner, it is desirable to measure it at a
laser intensity at which the number of the spots showing saturation of
the fluorescence intensity can be reduced as much as possible and the
entire fluorescence intensity can be raised. Such laser intensity, which
varies according to the test sample, can be determined by simple
examination.

[0074]The DNA microarray is not particularly limited, if it is a
microarray carrying all yeast genes, and, for example, "GeneChip"
manufactured by Affymetrix and "3D-Gene" manufactured by Toray Industries
Inc. can be used favorably. It is more preferably "3D-Gene."

[0075]Here, when the fluorescence intensity of all genes is determined by
using the DNA microarray, a gene showing a fluorescence intensity larger
by 5 times or more than the average will be defined as the "gene
expressed in a gene expression amount larger by 5 times or more than the
average relative expression amount of all genes." A gene showing a value
larger by 7 times or more will be defined as the "gene expressed in a
gene expression amount larger by 7 times or more than the average
relative expression amount of all genes." Further, a gene showing a value
larger by 10 times or more will be defined as the "gene expressed in a
gene expression amount larger by 10 times or more than the average
relative expression amount of all genes."

[0076]The promoter of a gene expressed in a gene expression amount larger
by 5 times or more than the average relative expression amount of all
genes after 50 hours from start of culture in continuous culture with
simultaneous filtration of a yeast strain having a lactic acid-producing
ability is the promoter of a gene that can be determined by DNA
microarray measurement by sampling the yeast strain at a point after 50
hours form start of culture in continuous culture with simultaneous
filtration of a yeast strain having a lactic acid-producing ability and
using the total RNAs extracted from the yeast.

[0077]The "promoter" means a nucleotide sequence involved in initiation of
transcription from gene to mRNA, and it normally indicates a sequence 5
terminal-sided upstream of the gene present on chromosome. The nucleotide
sequence length of the promoter is preferably 1 to 3000 bp, more
preferably 1 to 1000 bp, but it is not particularly limited, if it is a
nucleotide sequence that can initiate transcription of its downstream
gene into mRNA. The mutation and operation for improvement in
transcription activity of the promoter are already known, and the
promoters also include promoters modified by known methods.

[0079]More preferable are the promoters having a nucleotide sequence
selected from the following sequences (a) to (c): [0080](a) Promoters
having a nucleotide sequence represented by the following SEQ ID Nos. 1
to 3; [0081](b) Promoters having a nucleotide sequence that can hybridize
with a nucleotide sequence represented by the following SEQ ID Nos. 1 to
3 or a nucleotide sequence having part of it under stringent condition;
and [0082](c) Promoters having a nucleotide sequence obtained by
deletion, substitution and/or addition of one or more bases from a
nucleotide sequence selected from those represented by the following SEQ
ID Nos. 1 to 3:

[0083]The "stringent condition" is a condition in which a probe hybridizes
to its target sequence at a degree higher than that to other sequences
(e.g., at least twice larger than the background). The stringent
condition depends on the sequence and varies according to the environment
in which hybridization is carried out. Here, the stringent condition is a
hybridization temperature of 37° C. in the presence of 50%
formamide, and a more severe condition is a temperature of approximately
42° C. A further stringent condition is a hybridization
temperature of approximately 65° C. in the presence of formamide.

[0084]We provide a lactate dehydrogenase-expressing cassette containing
one of the promoters described above.

[0085]We also provide a lactate dehydrogenase-expressing cassette
containing a promoter selected from the following group (a) and a lactate
dehydrogenase-coding gene selected from the group (b):

(a) [0086](1) Promoters having a nucleotide sequence represented by any
one of SEQ ID Nos. 1 to 3; [0087](2) Promoters having a nucleotide
sequence that hybridizes with a nucleotide sequence represented by any
one of SEQ ID Nos. 1 to 3 or a nucleotide sequence having part of it
under stringent condition; and [0088](3) Promoters having a nucleotide
sequence obtained by deletion, substitution and/or addition of one or
more bases from the nucleotide sequence represented by any one of the
following SEQ ID Nos. 1 to 3; and(b) [0089](1) Genes coding lactate
dehydrogenase having a nucleotide sequence represented by any one of the
following SEQ ID Nos. 4 to 6; [0090](2) Genes coding lactate
dehydrogenase having a nucleotide sequence that hybridizes with a
nucleotide sequence represented by any one of the following SEQ ID Nos. 4
to 6 or a nucleotide sequence having part of it under stringent
condition; and [0091](3) Genes coding lactate dehydrogenase having a
nucleotide sequence obtained by deletion, substitution and/or addition of
one or more bases in the nucleotide sequences represented by any one of
the following SEQ ID Nos. 4 to 6:

[0092]We provide a transformant yeast strain having at least one lactate
dehydrogenase-expressing cassette in chromosome. The transformant yeast
strain can gave the lactate dehydrogenase-expressing cassette introduced
at any position of the chromosome where the LDH can be expressed from the
lactate dehydrogenase-expressing cassette. Preferably, it is a yeast in
which at least one gene selected from SED1 gene, CWP2 gene and EN01 gene
is substituted by the lactate dehydrogenase-expressing cassette.

[0093]The yeast can be used favorably, if it is a yeast into which the
lactate dehydrogenase-expressing cassette can be introduced. An example
thereof is a yeast belonging to Saccharomyces species,
Schizosaccharomyces species, Zygosaccharomyces species, Kluyveromyces
species or Candida species. It is preferably a yeast belonging to
Saccharomyces species. It is more preferably Saccharomyces cerevisiae.

Production Method of Lactic Acid

[0094]We provide a method of producing lactic acid, comprising culturing
the transformant yeast strain. The culture used may be batch culture,
feeding culture (fed batch culture), chemostat culture or continuous
culture. It is preferably continuous culture. It is more preferably
continuous culture of filtering the culture solution through a separation
membrane, recovering lactic acid from the filtrate, holding or feeding
back the unfiltered solution to the culture solution and replenishing the
medium to the culture solution.

[0095]Raw fermentation materials for the yeast are not particularly
limited, if they accelerate growth of the yeast in fermentation culture
and the desirable fermentation product lactic acid is produced
efficiently. A favorable fermentation raw material is a liquid medium
containing carbon sources, nitrogen sources, inorganic salts and, as
needed, amino acids and organic trace nutrients such as vitamins in
suitable amounts.

[0096]Examples of the carbon sources for use include sugars such as
glucose, sucrose, fructose, galactose and lactose; mixtures containing
these sugars such as starch hydrolysate, sweet potato syrup, sugar beet
syrup, Hi Test molasses and sugarcane extract; organic acids such as
acetic acid and fumaric acid; alcohols such as ethanol; glycerol; and the
like. The sugars above are carbohydrates having an aldehyde group or a
ketone group, which are the first oxidation products of polyvalent
alcohols, and they are grouped into aldoses having aldehyde groups and
ketoses having ketone groups.

[0099]When a particular nutrient is needed for growth of the yeast, the
nutrient can be added as a standard reagent or a natural product
containing the same. In addition, an antifoam may be added as needed.

[0100]The fermentation culture solution means a solution obtained after
growth of the yeast strain with the fermentation raw materials. The
composition of the fermentation raw materials to be added may be altered
properly according to the composition of the fermentation raw materials
used when the culture is initiated. If the composition of the
fermentation raw materials added is different from that of the
fermentation raw material added at first, modification leading to
increase in productivity of desirable lactic acid is preferable. It is
often possible to decrease the production cost of lactic acid, that is,
to increase the productivity of lactic acid in the broad sense, for
example by decreasing the percentages by weight of the nitrogen source,
inorganic salts, amino acids and organic trace amount nutrients such as
vitamins with respect to the carbon source. On the other hand, it may be
possible to improve the productivity of lactic acid, by increasing the
percentage by weight of nitrogen source, inorganic salts, amino acids and
organic trace-amount nutrients such as vitamins with respect to the
carbon source.

[0101]The concentrations of fermentation raw materials such as sugar in
the fermentation culture solution are preferably kept at 5 g/L or less.
The concentrations are kept more preferably at 3 g/L or less, more
preferably at 1 g/L or less. It is aimed at minimizing the loss of the
fermentation raw materials by withdrawal of the fermentation culture
solution. Thus, the concentrations of the fermentation raw materials in
the fermentation culture solution are desirably as low as possible.

[0102]Fermentation culture of yeast is normally, frequently carried out at
a pH of 3 to 8 and a temperature in the range of 20 to 40° C.
Because the product lactic acid is an acidic substance, the pH of the
fermentation culture solution is adjusted to a predetermined value in the
range above with an alkaline substance, urea, calcium carbonate, ammonia
gas or the like.

[0103]If the oxygen-supplying rate is desirably increased in the yeast
fermentation culture, it may be possible to increase the oxygen-supplying
rate for example, by means of keeping the oxygen concentration at 21% or
more by adding oxygen into air, pressurizing the fermentation culture
solution, raising the agitation velocity or raising the ventilation rate.
Alternatively if the oxygen-supplying rate is needed to be reduced, it
may be also possible to mix air with an oxygen-free gas such as carbon
dioxide, nitrogen or argon and supply the mixed gas.

[0104]Continuous culture (withdrawal) may be initiated after the yeast
concentration is increased by batch culture or fed batch culture in the
early phase of culture, or alternatively, continuous culture may be
started simultaneously with culture by seeding yeast at higher
concentration. It is possible to supply the fermentation raw material
solution and withdraw the cultured solution simultaneously from a
suitable point. The supply of the fermentation raw material and the
withdrawal of the cultured solution may not be carried out at the same
time. Alternatively, the supply of the fermentation raw material and the
withdrawal of the cultured solution may be carried out continuously or
intermittently. The aforedescribed nutrients needed for growth of the
microbes are preferably added to the fermentation raw material solution
for continuous growth of the microbes.

[0105]For more effective production, the concentration of the yeast in the
fermentation culture solution is preferably kept high in the range in
which the environment of the fermentation culture solution is not
inadequate for growth of the yeast and the death rate is not increased.
It is possible, for example, to obtain more favorable productivity by
keeping the yeast concentration, as dry weight, at 5 g/L or more. The
maximum concentration of the yeast is not particularly limited, if it
does not cause troubles in operation of the continuous fermentation
apparatus or lead to deterioration in productivity.

[0106]When continuous culture is carried out by using fresh microbes
capable of fermentation production, it is preferably, normally carried
out in a single fermentation reaction tank for control of culture.
However, in the case of a continuous culture method of producing lactic
acid with simultaneous growth of microbes, the number of the fermentation
reaction tanks is arbitrary. Multiple fermentation reaction tanks may be
used, for example because the capacity of a fermentation reaction tank is
small. It is possible in such a case to obtain the fermentation product
at high productivity, even if the continuous culture is carried out, as
the multiple fermentation reaction tanks are connected in parallel or in
series to each other by pipings.

[0107]Lactic acid contained in the culture solution produced by the method
of producing lactic acid may be separated and purified by combination of
known methods such as ion exchanging, concentration, distillation and
crystallization.

[0108]For example, a fermentation apparatus used in the method of
producing lactic acid is configured to have mainly a fermentation
reaction tank for production of lactic acid, holding the transformant
yeast strain therein, and a separation membrane element containing a
porous membrane for separation of the culture solution from the
transformant yeast strain by filtration. The separation membrane element
may be installed inside or outside the fermentation reaction tank.

[0109]In the method of producing lactic acid by continuous culture by
means of filtering the culture solution with a separation membrane,
recovering lactic acid from the filtrate, holding or feeding back the
unfiltered solution to the culture solution and adding the medium to the
culture solution, it is preferable to use a porous membrane having an
average micropore diameter of 0.01 μm or more and less than 1 μm as
the separation membrane and to filtrate the culture solution at a
filtration pressure, i.e., transmembrane pressure difference, in the
range of 0.1 to 20 kPa. Because it is not particularly necessary to
pressurize the fermentation reaction tank, a driving means for
circulation of the fermentation culture solution between the filtration
separation apparatus and the fermentation reaction tank is not needed,
and thus, the separation membrane element can be installed in the
fermentation reaction tank for reduction in size of the fermentation
culture apparatus.

[0110]The configuration of the porous membrane favorably used as the
separation membrane will be described below. The porous membrane is a
membrane having a suitable separability and water permeability according
to the quality and application of the treated water, and it is preferably
a porous membrane having a porous resin layer, from the points of
separation properties such as blocking performance, water permeability
and staining resistance. Such a porous membrane has a porous resin layer
functioning as a functional separation layer on the surface of the porous
base material. The porous base material strengthens the separation
membrane by supporting the porous resin layer.

[0111]The material for the porous base material is not particularly
limited and may be an organic or inorganic material, but an organic fiber
is used favorably. Examples of favorable porous base materials include
woven and nonwoven fabrics produced by using an organic fiber such as
cellulosic fiber, cellulose triacetate fiber, polyester fiber,
polypropylene fiber or polyethylene fiber, and, among the materials
above, nonwoven fabrics, which are cheap, produced easily and allow
relatively easier control of density, are used favorably.

[0112]The porous resin layer functions as a functional separation layer,
as described above, and an organic polymer membrane may be used
favorably. Examples of the materials for the organic polymer membrane
include polyethylene resins, polypropylene resins, polyvinyl chloride
resins, polyvinylidene fluoride resins, polysulfone resins, polyether
sulfone resins, polyacrylonitrile resins, cellulosic resins, cellulose
triacetate resins and the like. The organic polymer membrane may be a
mixture of resins containing the resin as principal component. The term
"principal component," as used herein, means that the component is
contained in an amount of 50 wt % or more, preferably 60 wt % or more. In
particular, the raw material constituting the porous resin layer is
preferably a resin allowing easy membrane formation as its solution that
is superior in physical durability and chemical resistance, such as
polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone
resin, polyether sulfone resin or polyacrylonitrile resin, and a
polyvinylidene fluoride resin or a resin containing it as the principal
component is used most favorably.

[0113]The polyvinylidene fluoride resin favorably used is a homopolymer of
vinylidene fluoride, but copolymers thereof with a vinyl monomer
copolymerizable with vinylidene fluoride are also used favorably.
Examples of the vinyl monomers copolymerizable with vinylidene fluoride
include tetrafluoroethylene, hexafluoropropylene, ethylene trichloride
fluoride, and the like.

[0114]The separation membrane may be a flat membrane or a hollow fiber
membrane. In the case of a flat membrane, the average thickness is
determined according to applications selection, but preferably selected
in the range of 20 μm or more and 5000 μm or less, more preferably
50 μm or more and 2000 μm or less.

[0115]As described above, the separation membrane is preferably a porous
membrane made of a porous base material and a porous resin layer. The
porous resin layer may or may not be penetrated into the porous base
material then, and the degree is selected according to application. The
average thickness of the porous base material is preferably selected in
the range of 50 μm or more and 3000 μm or less. When the porous
membrane is a hollow fiber membrane, the internal diameter of the hollow
fiber is preferably selected in the range of 200 μm or more and 5000
μm or less, and the film thickness is preferably selected in the range
of 20 μm or more and 2000 μm or less. The hollow fiber may contain
a tubular woven or knitted fabric of organic or inorganic fiber therein.

[0116]First, a method of producing a flat membrane, among the porous
membranes above, will be described briefly.

[0117]A film of a concentrated solution containing the resin and a solvent
is formed on the surface of a porous base material, while impregnation of
the concentrated solution into the porous base material is permitted.
Subsequently, only the film-sided surface of the film-carrying porous
base material is brought into contact with a coagulation bath containing
a nonsolvent, for solidification of the resin and formation of a porous
resin layer on the surface of the porous base material. The concentrated
solution is prepared by dissolving a resin in solvent. The concentrated
solution may contain a nonsolvent additionally. The temperature of the
concentrated solution is preferably selected normally in the range of 15
to 120° C., from the viewpoint of membrane-forming efficiency.

[0118]The concentrated solution may then contain a pore-forming agent
additionally. The pore-forming agent has an action to make the resin
layer porous, as it is extracted from the coated film when it is immersed
in the coagulation bath. It may control the size of the average micropore
diameter, by addition of the pore-forming agent. The pore-forming agent
is preferably highly soluble in the coagulation bath. Examples of the
pore-forming agents favorably used include inorganic salts such as
calcium chloride and calcium carbonate. Alternatively, a polyoxyalkylene
such as polyethylene glycol and polypropylene glycol; a water-soluble
polymer compound such as polyvinylalcohol, polyvinylbutyral and
polyacrylic acid; and glycerol may be used as the pore-forming agent.

[0119]The solvent is a compound dissolving resins. The solvent accelerates
formation of the porous resin layer in interaction with the resin and the
pore-forming agent. Examples of the solvents for use include
N-methylpyrrolidinone (NMP), N,N-dimethylacetamide (DMAc),
N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),
N-methyl-2-pyrrolidone, methylethyl-ketone, tetrahydrofuran,
tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone,
y-butylolactone, methylisoamylketone, dimethyl phthalate, propylene
glycol methylether, propylene carbonate, diacetone alcohol, glycerol
triacetate, acetone, methylethylketone and the like. Among them, solvents
in which the resin is highly soluble, such as NMP, DMAc, DMF and DMSO,
are used favorably. These solvents may be used alone or as a mixture of
two or more. The concentrated solution may be prepared by dissolving the
aforedescribed resin in the solvent described above, preferably at a
concentration of 5 wt % or more and 60 wt % or less.

[0120]For example, components other than solvents such as polyethylene
glycol, polyvinyl-alcohol, polyvinylpyrrolidone and glycerol may be added
to the solvent. The nonsolvent is a liquid not dissolving resins. The
nonsolvent controls the speed of resin solidification and thus the size
of the micropore. Water or an alcohol such as methanol or ethanol may be
used as the nonsolvent. In particular, water and methanol are preferable
from the point of price. The nonsolvent may be a mixture of these
solvents.

[0121]Hereinafter, the method of producing a hollow fiber membrane, among
the porous membranes, will be described briefly.

[0122]The hollow fiber membrane may be prepared by extruding a
concentrated solution containing a resin and a solvent out of the
external pipe of a double-pipe die and a hollow-forming fluid out of the
internal pipe of the double-pipe die and solidifying the solution by
cooling in a cooling bath.

[0123]The concentrated solution may be prepared by dissolving the resin
described in the method of producing a flat membrane in the solvent
described in the method of producing a flat membrane preferably at a
concentration of 20 wt % or more 60 wt % or less. The hollow-forming
fluid for use is normally gas or liquid. In addition, an additional
porous resin layer may be coated (laminated) on the outermost surface of
the hollow fiber membrane obtained. The lamination may be performed, for
example, for modification of the properties of the hollow fiber membrane,
such as hydrophilicity-hydrophobic and micropore diameter to desired
properties. The additional porous resin layer laminated may be prepared
by bringing the concentrated solution containing a resin dissolved in a
solvent into contact with a coagulation bath containing a nonsolvent and
thus solidifying the resin. For example, materials similar to those for
the organic polymer membrane described above may be used favorably as the
resin materials. As for the lamination method, the hollow fiber membrane
may be immersed into the concentrated solution or the concentrated
solution be applied on the surface of the hollow fiber membrane, and the
lamination amount may be adjusted after lamination by squeezing out part
of the concentrated solution adhered or blowing out the concentrated
solution with an air knife.

[0124]The separation membrane gives a separation membrane element in
combination with a supporting member. A separation membrane element
having a supporting plate used as the supporting member and a separation
membrane formed at least on one face of the supporting plate is a
favorable aspect of the separation membrane element having the separation
membrane. If it is difficult to increase the membrane area in the shape,
it is preferable to form a separation membrane on both faces of the
supporting plate for increase of water permeability.

[0125]When the porous membrane used as separation membrane has an average
micropore diameter in the range of 0.01 μm or more and less than 1
μm, as described above, the membrane shows both high exclusion rate
prohibiting leakage of microbes and sludge and high water permeability at
the same time, and it may retain favorable water permeability without
clogging for an extended period of time at higher accuracy and
reproducibility. If microbes are used, the average micropore diameter of
the porous membrane is preferably 0.4 μm or less, and the membrane can
be used more favorably, if its average micropore diameter is less than
0.2 μm. The water permeability may decrease when the average micropore
diameter is excessively smaller and, thus, the average micropore diameter
is 0.01 μm or more, preferably 0.02 μm or more and still more
preferably 0.04 μm or more.

[0126]The average micropore diameter may be determined by measuring the
diameters of all micropores observable in the range of 9.2
μm×10.4 μm by observation under scanning electron microscope
at a magnification of 10,000 times and averaging the diameters thus
obtained.

[0127]The standard deviation a of the average micropore diameter is
preferably 0.1 μm or less. In addition, it may obtain homogeneous
permeate, when the standard deviation of the average micropore diameter
is small, i.e., when the micropore diameter is uniform in size. The
standard deviation of the average micropore diameter is desirably smaller
as much as possible, because it is easier to control the fermentation
operation.

[0128]When the number of micropores observed in the range of 9.2
μm×10.4 μm described above is designated as N, each diameter
measured as Xk and the average of the micropore diameter as X(ave), the
standard deviation ρ of the average micropore diameter is calculated
according to the following Formula 1:

[0129]Permeability of the fermentation culture solution is one of the
important properties of the separation membrane, and pure water
permeability coefficient of the separation membrane before use may be
used as the indicator of permeability. The pure water permeability
coefficient of the separation membrane, as determined by using water
purified by reverse osmosis membrane at a temperature of 25° C.
and a head height of 1 m, is preferably 2×10-9
m3/(m2sPa). It may obtain practically sufficient water
permeability, when the pure water permeability coefficient is
2×10-9 m3/(m2sPa) or more and 6×10-7
m3/(m2sPa) or less. More preferable, the pure water
permeability coefficient is 2×10-9 m3/(m2sPa) or
more and 1×10-7 m3/(m2sPa) or less.

[0130]The membrane surface roughness of the separation membrane is a
factor exerting an influence on clogging of the separation membrane. It
may decrease the exfoliation coefficient and the film resistance of the
separation membrane favorably and carry out continuous fermentation at
lower transmembrane pressure difference, when the membrane surface
roughness is preferably 0.1 μm or less. It thus leads to prevention of
clogging and stabilized continuous fermentation, and for that reason, the
surface roughness is preferable lower as much as possible.

[0131]In addition, it would be possible, by reducing the membrane surface
roughness of the separation membrane, to reduce the shearing force
generated on the membrane surface during filtration of microbes, thus
leading to suppression of decomposition of the microbes and clogging of
the separation membrane, and to continue stabilized filtration for an
extended period of time.

[0132]The membrane surface roughness may be determined by using the
following atomic force microscope apparatus (AFM) and the following
apparatus under the condition below. The atomic force microscope
apparatus (AFM) used is not particularly limited, if it is an apparatus
equivalent to or higher than the following apparatus in grade.
[0133]Apparatus: atomic force microscope apparatus (Nanoscope IIIa,
manufactured by Digital Instruments Co., Ltd.) [0134]Condition: Probe:
SiN cantilever (manufactured by Digital Instruments Co., Ltd.)) [0135]:
Scan mode: contact mode (gas-phase measurement) underwater tapping mode
(underwater measurement) [0136]: Scanning range: 10 μm, 25 μm
square (gas-phase measurement) 5 μm, 10 μm square (underwater
measurement) [0137]: Scanning definition: 512×512 [0138]Sample
preparation: [0139]In measurement, the membrane sample was immersed in
ethanol at room temperature for 15 minutes and then in RO water for 24
hours and then dried in air before use. The RO water is water, from which
impurities such as ions and salts are removed by filtration with a
reverse osmosis membrane (RO membrane), a kind of filtration membrane.
The size of the pores in the RO membrane is approximately 2 nm or less.

[0140]The membrane surface roughness (drough) is calculated from the
height of each point in the Z axis direction observed under the AFM
according to the following Formula 2:

Formula 2 d rough = n = 1 N Z n - Z
_ N ( Formula 2 ) ##EQU00002##

[0141]Drough: Surface roughness (μm)

[0142]Zn: Height in Z axis (μm)

[0143] Z: Average height in the scanning range (μm)

[0144]N: Number of measured samples

[0145]The transmembrane pressure difference when microbes are filtered
through the separation membrane is not particularly limited, if it is a
condition preventing facile clogging by the microbes and the medium
components, but it is important to perform the filtration treatment at a
transmembrane pressure difference in the range of 0.1 kPa or more and 20
kPa or less. The transmembrane pressure difference is preferably in the
range of 0.1 kPa or more and 10 kPa or less, more preferably in the range
of 0.1 kPa or more and 5 kPa. Deviation form the range of the
transmembrane pressure difference may result in rapid clogging by the
microbes and the medium components, possibly leading to decrease of the
amount of permeate water and troubles in continuous fermentation
operation.

[0146]As for the driving force for filtration, the transmembrane pressure
difference may be generated in the separation membrane by the principle
of siphon of using the difference in liquid level (head difference)
between the fermentation culture solution and the separated
membrane-treated water. For filtration-driving force, a suction pump may
be installed to the side of separated membrane-treated water or a
pressurization pump may be installed to the fermentation culture solution
side of the separation membrane. The transmembrane pressure difference
may be controlled by modifying the difference in liquid level between the
fermentation culture solution and the separated membrane-treated water.
When a pump is used for generation of transmembrane pressure difference,
the transmembrane pressure difference may be controlled by the suction
pressure force and the transmembrane pressure difference may be
controlled by the gas or liquid pressure for pressurization of the
fermentation culture solution side. If such pressure control is needed,
it is possible to control the transmembrane pressure difference, by using
the pressure difference between the pressure of the fermentation culture
solution side and the pressure of the separated membrane-treated water
side as the transmembrane pressure difference.

[0147]In addition, the separation membrane preferably shows performance
allowing filtration treatment at a transmembrane pressure difference
during filtration treatment in the range of 0.1 kPa or more and 20 kPa or
less. As described above, the separation membrane preferably has a pure
water permeability coefficient before use, as calculated from the water
permeability that is determined by using reverse osmosis
membrane-purified water at a temperature of 25° C. and at a head
height of 1 m, preferably in the range of 2×10-9
m3/(m2sPa) or more, more preferably in the range of
2×10-9 m3/(m2sPa) or more and 6×10-7
m3/(m2sPa) or less.

[0148]A typical example of the continuous fermentation apparatus used in
the method of producing lactic acid, in which a separation membrane
element is installed in the fermentation reaction tank, is shown in FIG.
1. FIG. 1 is a schematic side view explaining the aspect of the
membrane-separation continuous fermentation apparatus. In FIG. 1, the
membrane-separation continuous fermentation apparatus essentially has a
fermentation reaction tank 1 for fermentation culture of yeast and a
head-difference control unit 3 for control of the amount of the
fermentation culture solution in the fermentation reaction tank 1. A
separation membrane element 2 is installed in the fermentation reaction
tank 1 and the separation membrane element 2 has a porous membrane
incorporated therein. Examples of the porous membranes for use include
the separation membrane and the separation membrane element disclosed in
WO 2002/064240 pamphlet.

[0149]Hereinafter, favorable aspects of the continuous fermentation by the
membrane-separation continuous fermentation apparatus shown in FIG. 1
will be described. The medium is fed into the fermentation reaction tank
1 continuously or intermittently by a medium-supplying pump 7. The medium
may be disinfected as needed by heating sterilization, heating
disinfection or filter sterilization treatment before supply into the
fermentation reaction tank 1. During fermentation production, the
fermentation solution in the fermentation reaction tank 1 may be agitated
as needed by a stirrer 5 in fermentation reaction tank 1. A desired gas
may be supplied, as needed, into the fermentation reaction tank 1 by a
gas-supplying apparatus 4. The gas supplied then may be recovered and
recycled back into the gas-supplying apparatus 4. In addition, the pH of
the fermentation solution in the fermentation reaction tank 1 may be
adjusted, as needed, by a pH sensor-control unit 9 and a pH-adjusted
solution supply pump 8. High-productivity fermentation production may
also be conducted by regulating, as needed, the temperature of the
fermentation culture solution in the fermentation reaction tank 1 by a
temperature controller 10.

[0150]Regulation of pH and temperature was exemplified here for regulation
of the physical and chemical conditions of the fermentation culture
solution by the instrumentation-control unit, but the regulation may also
be made, as needed, by a measurement of dissolved oxygen or ORP
(oxidation-reduction potential), and the physical and chemical conditions
may be regulated by using lactic acid concentration in the fermentation
culture solution, as determined by an analyzer such as online chemical
sensor, as an indicator. The method of supplying the medium continuously
or intermittently is not particularly limited, but the amount and
velocity of supplying the medium may be regulated properly by using the
measure values of physical and chemical environments of the fermentation
culture solution obtained by the instrumentation apparatus.

[0151]The fermentation culture solution are filtered and separated into
yeast and fermentation products by the separation membrane element 2
installed in the fermentation reaction tank 1 and the fermentation
products are discharged from the apparatus system. The filtered and
separated yeast remains in the apparatus system, as the yeast
concentration in the apparatus system is kept higher, thus permitting
high-productivity fermentation production. The filtration and separation
by the separation membrane element 2 is carried out, as driven by the
head pressure difference with the water surface in the fermentation
reaction tank 1, and does not demand an additional special driving force.
The filtration-separation speed of the separation membrane element 2 and
the amount of the fermentation solution in the fermentation reaction tank
1 may be regulated, as needed, properly with a level sensor 6 and a head
pressure difference control unit 3. Although filtration and separation by
separation membrane element 2 performed by head pressure difference was
shown as the aspect above, the filtration and separation may be
performed, as needed, by a pump or by suction filtration for example by
gas or liquid or pressurization of the apparatus system.

[0152]Hereinafter, a typical example of the fermentation apparatuses used
in the method of producing lactic acid, in which the separation membrane
element is installed outside the fermentation reaction tank, is shown in
the schematic view of FIG. 2. In FIG. 2 is a schematic side view
explaining another aspect of the membrane-separation continuous
fermentation apparatus.

[0153]In FIG. 2, the membrane-separation continuous fermentation apparatus
essentially has a fermentation reaction tank 1 for fermentation culture
of yeast, a membrane separation tank 12 having a separation membrane
element 2 therein that is connected to the fermentation reaction tank 1
via a fermentation culture solution-circulating pump 11 and a
head-difference control unit 3 for regulation of the amount of the
fermentation culture solution in the fermentation reaction tank 1. The
separation membrane element 2 has a porous membrane incorporated therein.
Examples of the porous membranes for use include the separation membrane
and the separation membrane element disclosed in WO 2002/064240 pamphlet.

[0154]In FIG. 2, the medium may be fed into the fermentation reaction tank
1 by a medium-supplying pump 7 and the fermentation culture solution in
the fermentation reaction tank 1 may be agitated, as needed, with a
stirrer 5. A desired gas may also be fed therein, as needed, by a
gas-supplying apparatus 4. The gas supplied may then be recovered and
recycled back into the gas-supplying apparatus 4. The pH of the
fermentation culture solution may be adjusted, as needed, with a pH
sensor-control unit 9 and a pH-adjusted solution supply pump 8. In
addition, the temperature of the fermentation culture solution may be
adjusted, as needed, by a temperature controller 10 for high-productivity
fermentation production. Further, the fermentation culture solution in
the apparatus is circulated between the fermentation reaction tank 1 and
the membrane separation tank 12 by a fermentation culture
solution-circulating pump 11. The fermentation culture solution
containing the fermentation products is filtered and separated into yeast
and fermentation products by the separation membrane element 2 and thus,
the fermentation product lactic acid may be discharged out of the
apparatus system.

[0155]The filtered and separated yeast remains in the apparatus system, as
the yeast concentration in the apparatus system is kept higher, thus
permitting high-productivity fermentation production. The filtration and
separation by the separation membrane element 2 may be carried out by the
head pressure difference from the water surface of the membrane
separation tank 12 and does not demand an additional special power. The
filtration-separation speed of the separation membrane element 2 and the
amount of the fermentation culture solution in the apparatus system may
be regulated, as needed, properly by a level sensor 6 and a head pressure
difference control unit 3. A desired gas may be supplied, as needed, into
the membrane separation tank 12 by the gas-supplying apparatus 4.

[0156]Although filtration and separation by separation membrane element 2
performed by head pressure difference was shown as the aspect above, the
filtration and separation may be performed, as needed, by a pump or by
suction filtration for example by gas or liquid or pressurization of the
apparatus system. The transmembrane pressure difference may be adjusted
and controlled by the means above.

[0157]Hereinafter, the separation membrane and the separation membrane
element disclosed in WO 2002/064240 pamphlet, a favorable separation
membrane element, will be described briefly with reference to a drawing.
FIG. 3 is schematic perspective view explaining an aspect of the
separation membrane element.

[0158]The separation membrane element, as shown in FIG. 3, has a rigid
supporting plate 13 and a channel material 14 and the separation membrane
15 described above formed in that order on both faces thereof. The
supporting plate 13 has dents 16 on both faces. The separation membrane
15 allows filtration of the fermentation culture solution. The channel
material 14 is a material for transport of the filtrate from the
separation membrane 15 efficiently to the supporting plate 13. The
filtrate fed to the supporting plate 13 advances through the dent 16 of
the supporting plate 13 into the water-collecting pipe 17 and is then
discharged out of the continuous fermentation apparatus. A method such as
head pressure difference, pump, suction filtration for example with
liquid or gas, or pressurization of the apparatus system may be used as
the power for discharging the filtrate.

[0159]FIG. 4 is a schematic perspective view explaining another separation
membrane element. The separation membrane element, as shown in FIG. 4,
mainly has a bundle of hollow-fiber separation membranes 18 (porous
membranes), a top resin-sealing layer 19 and a bottom resin-sealing layer
20. The separation membranes 18 are adhered and fixed to each other in
the bundle shape by the top resin-sealing layer 19 and the bottom
resin-sealing layer 20. The hollow regions of the hollow fiber membranes
(porous membranes) of the separation membrane bundle 18 are sealed with
the bottom resin-sealing layer 20 by adhesion and fixation in a structure
preventing leakage of the culture solution. On the other hand, the top
resin-sealing layer 19 does not seal the internal pores of the hollow
fiber membranes (porous membranes) of the separation membrane bundle 18,
as it is formed in a structure allowing flow of the filtrate into the
water-collecting pipe 22. The separation membrane element may be
installed in the continuous fermentation apparatus via the supporting
frame 21. The fermentation product-containing filtrate filtered through
the separation membrane bundle 18 advances through the hollow region of
the hollow fiber membranes and the water-collecting pipe 22 and is
discharged out of the continuous fermentation apparatus. A method such as
head pressure difference, pump, suction filtration for example with
liquid or gas or pressurization of the apparatus system may be used as
the power for discharging the filtrate.

[0160]The member for the separation membrane element of the continuous
fermentation apparatus used in the method of producing lactic acid is
preferably a member resistant to high-pressure steam sterilization
operation. If the continuous fermentation apparatus is sterilizable
internally, it becomes possible to avoid the danger of undesired
microbial contamination during continuous fermentation and perform more
stabilized continuous fermentation. The member constituting the
separation membrane element is preferably resistant to the condition of
high-pressure steam sterilization operation, specifically at 121°
C. for 15 minutes. For example, a metal such as stainless steel or
aluminum or a resin such as polyamide resin, fluorine resin,
polycarbonate resin, polyacetal resin, polybutylene terephthalate resin,
PVDF, modified polyphenylene ether resin or polysulfone resin may be
selected favorably for the separation membrane element member.

[0161]In the continuous fermentation apparatus used in the method of
producing lactic acid, the separation membrane element may be installed
in the fermentation reaction tank, as shown in FIG. 1, or outside the
fermentation reaction tank, as shown in FIG. 2. If the separation
membrane element is installed outside the fermentation reaction tank, a
membrane separation tank may be installed separately and the separation
membrane element installed therein, and the culture solution may be
filtered continuously through the separation membrane element, as the
culture solution is circulated between the fermentation reaction tank and
the membrane separation tank.

[0162]In the continuous fermentation apparatus used in the method of
producing lactic acid, the membrane separation tank is desirably
sterilizable with high-pressure steam. If the membrane separation tank is
sterilizable with high-pressure steam, contamination by undesired
bacteria may be avoided easily.

[0163]Continuous fermentation, if carried out by the method of producing
lactic acid by continuous fermentation, gives higher volumetric
production rate and enables extremely efficient fermentation production,
compared to batchwise fermentation. The fermentation production rate by
continuous culture may be calculated according to the following Formula
3:

[0164]The fermentation production rate by batchwise culture may be
calculated by dividing the amount of the product generated when the raw
carbon source is all consumed (g) by the period needed for consumption of
the carbon source (h) and the volume of the fermentation culture solution
at the time (L).

[0165]The continuous culture is carried out, as it is stabilized, without
deterioration in lactic acid yield and production rate for an extended
period of time, by culturing a yeast having a lactate
dehydrogenase-expressing cassette containing the promoter of a gene
showing a gene expression amount larger by 5 times or more, preferably 7
times or more and more preferably 10 times or more, than the average
relative expression amount of all genes after 50 hours from start of
culture while filtering the yeast through a separation membrane, and
thus, the continuous culture is preferably continued at least for 100
hours or more, preferably for 200 hours or more and more preferably for
300 hours or more.

[0166]Both D- and L-lactic acids may be provided.

[0167]Lactic acid obtained by the method of producing lactic acid may be
provided mainly as a raw material for polylactic acid.

EXAMPLES

[0168]Hereinafter, favorable aspects will be described with reference to
Examples, but it should be understood that this disclosure is not
restricted at all by these Examples.

Reference Example 1

Preparation of Yeast Strain Having Lactic Acid-Producing Ability

[0169]A yeast containing the Xenopus laevis-derived ldh gene having the
nucleotide sequence shown by SEQ ID No. 4 at a site downstream of PDC1
promoter was used as the yeast strain having a lactic acid-producing
ability. The Xenopus laevis-derived ldh gene was cloned by PCR method. A
phagemid DNA prepared from Xenopus laevis kideny-derived cDNA library
(manufactured by STRATAGENE) according to the attached protocol was used
as a template in PCR.

[0170]KOD-Plus polymerase (manufactured by Toyobo) was used for PCR
amplification reaction, and the reaction buffer, dNTP mix and others used
were those attached to the kit. 50 μL, of a reaction system containing
50 ng/sample of the phagemid DNA prepared according to the attached
protocol as described above, 50 pmol/sample of primers and 1 unit/sample
of KOD-Plus polymerase was prepared. The reaction solution was denatured
at a temperature of 94° C. for 5 minutes in a PCR amplification
system iCycler (manufactured by BIO-RAD), and then subjected to 30 cycles
of heat denaturation at 94° C. for 30 seconds, primer annealing at
55° C. for 30 seconds and extension of complementary chain at
68° C. for 1 minute, and then cooled to a temperature of 4°
C. The gene amplification primers (SEQ ID Nos. 7 and 8) were prepared so
that a SalI-recognizing sequence is added to the 5 terminal side and a
NotI-recognizing sequence to the 3 terminal side.

[0171]The PCR amplification fragments were purified, and the terminals
thereof were phosphorylated by T4 polynucleotide Kinase (manufactured by
Takara Bio Inc.) and then ligated to pUC118 vector (which was previously
cleaved with a restriction enzyme HincII and had the cut surface
dephosphorylated). Ligation was carried out by using a DNA ligation kit
Ver.2 (manufactured by Takara Bio Inc.). The ligation solution was
transformed into competent cells of E. coli DH5α (manufactured by
Takara Bio Inc.) and the mixture was seeded and cultured overnight on a
LB plate containing an antibiotic ampicillin at a concentration of 50
μg/mL. Plasmid DNAs in the colonies obtained were collected by
Miniprep and cleaved with restriction enzymes SalI and NotI, and plasmids
containing the inserted Xenopus laevis-derived ldh gene were selected.
All of the series of operations were carried out according to the
attached protocol.

[0172]The pUC118 vectors containing the inserted Xenopus laevis-derived
ldh gene were cleaved by restriction enzymes SalI and NotI; the DNA
fragments were separated by 1% agarose gel electrophoresis; and the
fragments containing the Xenopus laevis-derived ldh gene were purified by
a common method. The fragments containing the ldh gene were ligated to
the XhoI/NotI cleavage sites of the expression vector pTRS11 shown in
FIG. 5; the plasmid DNAs were recovered by a method similar to that
above; and an expression vector containing the inserted Xenopus
laevis-derived ldh gene was selected by cleaving the expression vectors
with restriction enzymes XhoI and NotI. Hereinafter, the expression
vector containing the inserted Xenopus laevis-derived ldh gene thus
prepared will be designated as pTRS102.

[0173]A 1.3 kb PCR fragment containing the Xenopus laevis-derived ldh gene
and a TDH3 terminator sequence was amplified, by PCR using pTRS102 as
amplification template and oligonucleotides (SEQ ID Nos. 9 and 10) as
primer set. The oligonucleotide of SEQ ID No. 9 was designed to have a
sequence corresponding to the 60 by sequence upstream of the start codon
of PDC1 gene.

[0174]Then, a 1.2 kb PCR fragment containing a yeast selectable marker
TRP1 gene was amplified by PCR using plasmid pRS424 as amplification
template and oligonucleotides (SEQ ID Nos. 11 and 12) as primer set. The
oligonucleotide of SEQ ID No. 12 was designed to have an added sequence
corresponding to the 60 by sequence downstream from the termination codon
of PDC1 gene.

[0175]Each DNA fragment was isolated by 1% agarose gel electrophoresis and
purified according to a common method. By PCR using a mixture of the 1.3
kb fragment and the 1.2 kb fragment thus obtained as amplification
templates and oligonucleotides (SEQ ID Nos. 9 and 12) as primer set, an
approximately 2.5 kb PCR fragment having sequences equivalent to the
upstream and downstream 60 by sequences of PDC 1 gene respectively at the
5 and 3 terminals and containing Xenopus laevis-derived ldh gene, TDH3
terminator and TRP1 gene connected thereto was amplified.

[0176]The PCR fragments were isolated by 1% agarose gel electrophoresis
and purified according to a common method; they are transformed into a
yeast Saccharomyces cerevisiae NBRC10505 strain; and a transformant
having the Xenopus laevis-derived ldh gene introduced on chromosome at a
site downstream of the PDC1 gene promoter was selected by culture in a
tryptophan-free medium.

[0177]The fact that the transformant thus obtained is a yeast having a
Xenopus laevis-derived ldh gene introduced at a site downstream of the
PDC 1 gene promoter on chromosome was confirmed in the following manner:
First, the genome DNA of the transformant was prepared with a genome DNA
extraction kit Gentorukun (manufactured by Takara Bio Inc.) and
production of amplified DNA fragment of approximately 2.8 kb was
confirmed by PCR using the prepared genome DNA of the transformant as an
amplification template and oligonucleotides (SEQ ID Nos. 12 and 13) as
primer set. On the other hand, nontransformants gave amplified DNA
fragments of approximately 2.1 kb by the PCR. Hereinafter, the
transformant having the Xenopus laevis-derived ldh gene introduced at a
site downstream of the PDC1 gene promoter on chromosome will be referred
to as strain B2. The upstream and downstream sequences of the PDC1 gene
can be obtained from the Saccharomyces Genome Database
(www.yeastgenome.org).

TABLE-US-00006
SEQ ID No. 13:
caaatatcgt ttgaatattt ttccg

[0178]Subsequently, the yeast strain SW015 described in WO 2007/043253
pamphlet, in which the pdc 1 gene is replaced with the TRP1 marker and
the pdc5 gene has a temperature-sensitive mutation, and the strain B2
obtained above were conjugated, to give a diploid cell. The diploid cell
was forced to form ascus in an ascus-forming medium. The ascus was
dissected with a micromanipulator; respective haploid cells were
collected; and nutritional requirements of respective haploid cells were
studied. Among the haploid cells obtained a strain having the Xenopus
laevis-derived ldh gene inserted into the pdc1 gene locus and having a
temperature-sensitive mutation in the pdc5 gene (not viable at 34°
C.) was selected. The yeast strain was designated as strain SU014.

[0192]The optical purity of L-lactic acid is calculated according to the
following Formula:

Optical purity (%)=100×(L-D)/(L+D)

Herein, L represents the concentration of L-lactic acid, and D represents
the concentration of D-lactic acid.

[0193]L-Lactic acid was detected and D-lactic acid was detected only in an
amount of less than detection limit by HPLC analysis. The results above
demonstrate that the strain SU014 has L-lactic acid-producing ability.

Reference Example 2

Method of Preparing Separation Membrane

[0194]A porous membrane prepared by the following method was used as the
separation membrane.

[0195]A polyvinylidene fluoride (PVDF) resin was used as the resin, a
polyethylene glycol (PEG) having a molecular weight of approximately
20,000 as the pore-forming agent, N,N-dimethylacetamide (DMAc) as the
solvent and pure water as the nonsolvent, and these components were mixed
under thorough agitation at a temperature of 90° C., to give a
concentrated solution in the following composition: [0196]PVDF: 13.0 wt
% [0197]PEG: 5.5 wt % [0198]DMAc: 78.0 wt % [0199]Pure water: 3.5 wt %

[0200]The concentrated solution was cooled to a temperature of 25°
C. and then coated on a polyester fiber nonwoven fabric (porous base
material) having a density of 0.48 g/cm3 and a thickness of 220
μm, and the resulting nonwoven fabric was immersed, immediately after
application, in pure water at a temperature of 25° C. for 5
minutes and thrice in hot water at a temperature of 80° C. for
wash out of DMAc and PEG, to give a porous membrane (separation membrane)
having a porous resin layer. A range of 9.2 μm×10.4 μm on the
surface of the porous resin layer on the side where the concentrated
solution for separation membrane was coated was observed under scanning
electron microscope at a magnification of 10,000 times, to show that the
average diameter of all micropores observed was 0.02 μm. The pure
water permeability of the separation membrane evaluated was
2×10-9 m3/(m2sPa). The water permeability was
determined at a head height of 1 m, by using RO membrane-purified water
at a temperature of 25° C. The standard deviation of the average
micropore diameter was 0.0055 μm and the membrane surface roughness
was 0.1 μm.

Reference Example 3

Filtration Continuous Culture of the Yeast Strain Having a Lactic
Acid-Producing Ability by Using a Separation Membrane

[0201]Filtration continuous culture was carried out by using the strain
SU014 prepared in Reference Example 1 and the continuous culture
apparatus shown in FIG. 1. A lactic acid fermentation medium in the
composition shown in Table 1 was used as the medium. The medium was
sterilized with high-pressure (2 atm) steam at a temperature of
121° C. for 15 minutes before use. Stainless steel and a
polysulfone resin molding were used for the separation membrane element
members and the porous membrane prepared in Reference Example 2 was used
as the separation membrane. The operational condition in the present
Example is as follows, unless specified otherwise: [0202]Volume of
reaction tank volume: 2 (L) [0203]Volume of fermentation reaction tank:
1.5 (L) [0204]Separation membrane used: PVDF filtration membrane
(prepared in Reference Example 2) [0205]Effective filtration area of
membrane separation element: 120 cm2[0206]Temperature adjusted to:
30 (° C.) [0207]Ventilation rate of fermentation reaction tank:
air: 0.01 (L/min), nitrogen gas: 0.19 (L/min) [0208]Agitation velocity of
fermentation reaction tank: 800 (rpm) [0209]pH adjustment: adjusted to pH
5 with 1N NaOH [0210]Antifoam: a sterilized antifoam PE-L (manufactured
by Wako Pure Chemical Industries) added in an amount of 200 μL every 3
hours. [0211]Sterilization: culture tank containing a separation membrane
element and the medium used were all sterilized with high-pressure steam
in an autoclave at 121° C. for 20 min.

[0212]First, strain SU014 was cultured as shaken in a test tube containing
10 ml of a lactic acid fermentation medium overnight (preprepreculture).
The culture solution obtained was transferred into 100 ml of a fresh
lactic acid fermentation medium and the mixture was cultured as shaken in
a 500-ml Sakaguchi flask for 24 hours at 30° C. (prepreculture).
The prepreculture solution was seeded into 1.5 L of a lactic acid
fermentation medium (glucose concentration: 70 g/L) placed in the
continuous culture apparatus shown in FIG. 1, and the mixture was
cultured for 24 hours, while the reaction tank 1 was agitated with an
attached stirrer 5 at 400 rpm, as the ventilation rate, the temperature
and the pH of the reaction tank 1 were adjusted (preculture). Immediately
after the preculture, the lactic acid fermentation medium was supplied
thereto continuously, and lactic acid was produced by continuous culture,
while the membrane permeation amount was controlled to keep the
fermentation solution amount in the continuous culture apparatus constant
at 1.5 L. The concentrations of lactic acid produced and residual glucose
in the membrane-permeated fermentation solution were determined as
needed. The lactic acid/sugar yield and the lactic acid production rate
were calculated from the amounts of generated lactic acid and the
supplied raw glucose that was calculated from the glucose concentration.
The glucose concentration was determined by using "Glucose Test Wako C"
(registered trade name) (manufactured by Wako Pure Chemical Industries).
As a result, it was found that, although the continuous culture continued
consistently for a period of up to 200 hours, the lactic acid/sugar yield
and the lactic acid production rate declined after about 200 hours,
because of decrease of the concentration of lactic acid accumulated.

Example 1

Fluctuation of Gene Expression During Continuous Culture

[0213]For evaluation of fluctuation in gene expression during the
continuous culture in Reference Example 3, the culture solution samples
were obtained after 70 and 210 hours from the start of the continuous
culture of Reference Example 3 and the total RNAs therein were extracted
from the yeast. In extraction of the total RNA, the yeast obtained was
suspended in 10 ml of a buffer solution for homogenization (50 mM sodium
acetate (pH 5.3), 10 mM EDTA DEPC treated) to an OD600 of 0.2 and
the mixture was transferred into a 50 ml tube. 500 μL of 20% SDS was
added thereto; 12 ml of phenol (saturated with homogenization buffer
solution) previously warmed at 65° C. was added additionally
thereto; and the mixture was agitated with a Voltex mixer for 5 seconds.
The mixture was kept at 65° C. for 4 minutes and then cooled
rapidly to room temperature in a dry ice/ethanol bath. It was centrifuged
at room temperature (5 min. 12000 G); the aqueous supernatant was
transferred into a separate 50 ml tube; and PCI (pH 5.3) in the same
amount was added thereto for extraction. The aqueous supernatant was
transferred into a separate 50 ml tube and chloroform in the same amount
was added for extraction. The aqueous supernatant was transferred into a
separate 50 ml tube; 3 M sodium acetate (pH 5.3) in 1/10 amount was
added; and the mixture was subjected to ethanol precipitation. The
resulting pellets obtained were washed twice with 80% ethanol, dried to
solidness and dissolved in RNA-free water, to give a total RNA sample.

[0214]The concentration of the total RNA sample obtained was determined
with an absorptiometer and 1 μg of the sample was used for DNA
microarray analysis. The DNA microarray was carried out by using
"3D-Gene" manufactured by Toray Industries Inc. according to the attached
protocol. The scanner used was ScanArray Express (PerkinElmer), and
scanning was performed under the condition of Cyanine 5 (PMT value: 55%)
(for measurement of average fluorescence intensity after 210 hours of
culture) or Cyanine 3 (PMT value: 70%) (for measurement of average
fluorescence intensity after 70 hours of culture), to give an image. The
fluorescence intensity of each spot in the image obtained was digitalized
by GenePix Pro 5.0 software (Axon Instruments), and the median value was
calculated. Then, the average fluorescence intensity of all spots in each
sample was calculated.

[0216]The results identified the genes that are expressed in an amount
larger by 10 times or more than the average relative expression amount of
all genes after 50 hours from start of culture in continuous culture with
simultaneous filtration of a yeast strain having a lactic acid-producing
ability.

Example 2

Introduction of ldh Gene into SED1, CWP2 and ENO1 Gene Locuses

[0217]Based on the results obtained in Example 1, the ldh gene shown by
SEQ ID No. 4 was introduced into the SED1 gene, CWP2 gene and ENO1 gene
locuses.

[0218]In introduction thereof into the SED1 gene locus, a 1.3 kb PCR
fragment containing the Xenopus laevis-derived ldh gene and the TDH3
terminator sequence was amplified by PCR using pTRS102 prepared in
Reference Example 1 as amplification template and the oligonucleotides
(SEQ ID Nos. 10 and 14) as primer set. The oligonucleotide of SEQ ID No.
14 was designed to add a sequence corresponding to the 60 by sequence
upstream of the start codon of SED1 gene.

[0219]Then, an approximately 1.3 kb PCR fragment containing a yeast
selectable marker HIS3 gene was amplified by PCR using the plasmid pRS423
as amplification template and oligonucleotides (SEQ ID Nos. 11 and 15) as
primer set. The oligonucleotide of SEQ ID No. 15 was designed to add a
sequence corresponding to the 60 by sequence downstream the termination
codon of SED1 gene.

[0220]Each DNA fragment was separated by 1% agarose gel electrophoresis
and purified by a common method. An approximately 2.6 kb PCR fragment
having connected the Xenopus laevis-derived ldh gene, the TDH3 terminator
and the HIS3 gene having added the sequences corresponding to the
upstream and downstream 60-bp sequences of the SED1 gene respectively at
the 5 and 3 terminals was amplified, by PCR using a mixture of two kinds
of approximately 1.3 kb fragments thus obtained as amplification template
and oligonucleotides (SEQ ID Nos. 14 and 15) as primer set.

[0221]The PCR fragment was separated by 1% agarose gel electrophoresis and
purified by a common method, and then transformed into strain SU014, and
a transformant having the Xenopus laevis-derived ldh gene incorporated at
a site downstream of the SED1 gene promoter on chromosome was selected by
culture in a histidine-free medium.

[0222]The fact that the transformant thus obtained is a yeast having the
Xenopus laevis-derived ldh gene incorporated at a site downstream of the
SED1 gene promoter on chromosome was confirmed in the following manner:
First, the genome DNA of the transformant was prepared by using a genome
DNA extraction kit Gentorukun (manufactured by Takara Bio Inc.) and
production of an approximately 2.9 kb amplification DNA fragment was
confirmed by PCR using it as amplification template and oligonucleotides
(SEQ ID Nos. 16 and 17) as primer set. Nontransformants give an
approximately 1.4 kb amplification DNA fragment by the PCR. Hereinafter,
the transformant having the Xenopus laevis-derived ldh gene incorporated
at a site downstream of the SED1 gene promoter on chromosome will be
referred to as strain SU015.

[0223]Subsequently, in introduction into the CWP2 gene locus, the 1.3 kb
PCR fragment containing the Xenopus laevis-derived ldh gene and the
TDH3terminator sequence was amplified, by PCR using the pTRS102 prepared
in Reference Example 1 as amplification template and oligonucleotides
(SEQ ID Nos. 10 and 18) as primer set. The oligonucleotide of SEQ ID No.
21 was designed to add a sequence corresponding to the 60 by sequence
upstream of the start codon of CWP2 gene.

[0224]Subsequently, the 1.3 kb PCR fragment containing a yeast selectable
marker HIS3 gene was amplified by PCR using the plasmid pRS423 as
amplification template and oligonucleotides (SEQ ID Nos. 11 and 19) as
primer set. The oligonucleotide of SEQ ID No. 19 was designed to add a
sequence corresponding to the 60 by sequence downstream of the
termination codon of CWP2 gene.

[0225]Each DNA fragment was separated by 1% agarose gel electrophoresis
and purified by a common method. The approximately 2.6 kb PCR fragment
having connected the Xenopus laevis-derived ldh gene, the TDH3 terminator
and the HIS3 gene having added sequences corresponding to the 60 by
sequences upstream and downstream of the CWP2 gene respectively at the 5
and 3 terminals was amplified, by PCR using a mixture of the two kinds of
approximately 1.3 kb fragments as amplification template and
oligonucleotides (SEQ ID Nos. 18 and 19) as primer set.

[0226]The PCR fragment was separated by 1% agarose gel electrophoresis and
purified by a common method; it is transformed into strain SU014; and a
transformant strain having the Xenopus laevis-derived ldh gene
incorporated at a site downstream of the CWP2 gene promoter on chromosome
was selected by culture in a histidine-free medium.

[0227]The fact that the transformant thus obtained is a yeast having the
Xenopus laevis-derived ldh gene incorporated at a site downstream of the
CWP2 gene promoter on chromosome was confirmed in the following manner:
First, the genome DNA of the transformant was prepared by using a genome
DNA extraction kit Gentorukun (manufactured by Takara Bio Inc.), and it
was found by PCR using it as an amplification template and
oligonucleotides (SEQ ID Nos. 20 and 21) as primer set that an
approximately 2.9 kb amplification DNA fragment was obtained.
Nontransformants give an approximately 0.7 kb amplification DNA fragment
by the PCR. Hereinafter, the transformant having the Xenopus
laevis-derived ldh gene at a site downstream of the CWP2 gene promoter on
chromosome will be referred to as strain SU016.

[0228]Subsequently, in introduction into the ENO1 gene locus, a 1.3 kb PCR
fragment containing the Xenopus laevis-derived ldh gene and the TDH3
terminator sequence was amplified, by PCR using the pTRS102 prepared in
Reference Example 1 as amplification template and oligonucleotides (SEQ
ID Nos. 10 and 22) as primer. The oligonucleotide of SEQ ID No. 22 was
designed to add a sequence corresponding to the 60 by sequence upstream
of the start codon of ENO1 gene.

[0229]Subsequently, an approximately 1.3 kb PCR fragment containing a
yeast selectable marker URA3 gene was amplified by PCR using the plasmid
pRS426 as amplification template and oligonucleotides (SEQ ID Nos. 11 and
23) as primer set. The oligonucleotide of SEQ ID No. 23 was designed to
add a sequence corresponding to the 60 by sequence downstream of the
termination codon of ENO1 gene.

[0230]Each DNA fragment was separated by 1% agarose gel electrophoresis
and purified by a common method. An approximately 2.6 kb PCR fragment
having connected the Xenopus laevis-derived ldh gene, the TDH3 terminator
and the URA3 gene having added sequences corresponding to the 60 by
sequences upstream and downstream of EN01 gene respectively at the 5 and
3 terminals was amplified, by PCR using a mixture of the two kinds of 1.3
kb fragments obtained as an amplification template and oligonucleotides
(SEQ ID Nos. 22 and 23) as primer set.

[0231]The PCR fragment was separated by 1% agarose gel electrophoresis and
purified by a common method, and then transformed into the strain SU014,
and a transformant having the Xenopus laevis-derived ldh gene
incorporated at a site downstream of the promoter of ENO 1 gene on
chromosome was selected by culture in an uracil-free medium.

[0232]The fact that the transformant thus obtained is a yeast having the
Xenopus laevis-derived ldh gene incorporated at a site downstream of the
promoter of EN01 gene on chromosome was confirmed in the following
manner: First, the genome DNA of the transformant was prepared by using a
genome DNA extraction kit Gentorukun (manufactured by Takara Bio Inc.),
and an approximately 2.9 kb amplification DNA fragment was found to be
prepared by PCR using it as amplification template and oligonucleotides
(SEQ ID Nos. 24 and 25) as primer set. Nontransformants give an
approximately 1.7 kb amplification DNA fragment by the PCR. Hereinafter,
the transformant having the Xenopus laevis-derived ldh gene incorporated
at a site downstream of the ENO1 gene on chromosome will be referred to
as strain SU017.

[0233]Subsequently, the Xenopus laevis-derived ldh gene was introduced
into the ENOL gene locus of strain SU015. Introduction of the Xenopus
laevis-derived ldh gene into the EN01 gene locus of strain SU015 and
confirmation thereof were carried out in a manner similar to the method
described above, for preparation of the strain SU017, except that the
strain SU014 was replaced with the strain SU015. The transformant
obtained will be referred to as strain SU018.

[0235]Then, the human- and Leuconostoc mesenteroides-derived ldh genes
shown by SEQ ID Nos. 5 and 6 were cloned. First, the method of cloning
the human-derived ldh gene will be described below: [0236]A human
breast cancer cell line (MCF-7) was cultured and recovered; the total RNA
thereof was extracted by using TRIZOL Reagent (manufactured by
Invitrogen); and cDNAs were prepared in reverse transcription reaction by
using SuperScript Choice System (manufactured by Invitrogen) using the
total RNA as template. These operations were performed in detail
respectively according to the attached protocols. The cDNAs obtained were
used as amplification templates in subsequent PCR.

[0237]The ldh gene was cloned by PCR by KOD-Plus-polymerase using the
cDNAs obtained by the operations as amplification template and the
oligonucleotides of SEQ ID Nos. 26 and 27 as primer set. Each PCR
amplification fragment was purified; the terminal thereof was
phosphorylated by T4 polynucleotide kinase (manufactured by Takara Shuzo
Co., Ltd.) and ligated to a pUC118 vector (previously cleaved with
restriction enzyme HincII and the cut surface dephosphorylated). The
ligation was carried out by using a DNA ligation kit Ver.2 (manufactured
by Takara Shuzo Co., Ltd.). A plasmid containing a subcloned
human-derived ldh gene (accession number; AY009108, SEQ ID No. 5) was
obtained by transforming E. coli DH5α with the ligated plasmid
product and recovering the plasmid DNA. The pUC118 plasmid having the
obtained ldh gene inserted therein was digested with restriction enzymes
XhoI and NotI, and each DNA fragment obtained was inserted into a yeast
expression vector pTRS11 at the XhoI/NotI cleavage sites, to give a
human-derived ldh gene-expressing plasmid pTRS48.

[0238]Hereinafter, the method of cloning the Leuconostoc
mesenteroides-derived ldh gene will be described.

[0239]The Leuconostoc mesenteroides-derived ldh gene was cloned by gene
total synthesis to which the PCR method is applied, with reference to the
sequence (SEQ ID No. 6) described in Res. Microbiol, 146, 291-302 (1995).
The XhoI-recognizing sequence was added to the 5 terminal side and the
NotI-recognizing sequence to the 3 terminal side during the total
synthesis, and the PCR fragment was TA-cloned into the pTA2 vector.
Ligation was carried out by using a DNA ligation kit Ver.2 (manufactured
by Takara Shuzo Co., Ltd.). A plasmid having the subcloned Leuconostoc
mesenteroides-derived ldh gene (SEQ ID No. 6) was obtained by
transforming E. coli DH5α with the ligation plasmid product and
recovering the plasmid DNA. The pTA2 plasmid containing the inserted ldh
gene obtained was digested with restriction enzymes XhoI and NotI, and
each DNA fragment obtained was inserted into the yeast expression vector
pTRS11 at the XhoI/NotI cleavage site, to give a Leuconostoc
mesenteroides-derived ldh gene-expressing plasmid pTRS152.

[0240]Subsequently, the ldh genes of SEQ ID Nos. 5 and 6 were introduced
into the locuses of PDC1 gene, SED1 gene, CWP2 gene and ENO1 gene.

[0241]By PCR using plasmid pTRS48 and pTRS152 as amplification templates
and the oligonucleotides of SEQ ID Nos. 28 and 10 (pTRS48) and SEQ ID
Nos. 29 and 10 (pTRS152) as primer sets, a DNA fragment having the
terminator sequences of 1.3 kb human-derived ldh gene and Saccharomyces
cerevisiae-derived TDH3 gene and a DNA fragment containing the terminator
sequences of Leuconostoc mesenteroides-derived ldh gene and the
Saccharomyces cerevisiae-derived TDH3 gene were amplified. Also by PCR
using the plasmid pRS424 as amplification template and the
oligonucleotides of SEQ ID Nos. 11 and 12 as primer set, a DNA fragment
containing a 1.2 kb Saccharomyces cerevisiae-derived TRP1 gene was
amplified. Each DNA fragment was separated by 1.5% agarose gel
electrophoresis and purified by a common method. Each of the products
obtained by PCR using a mixture of the 1.3 kb fragment and the 1.2 kb
fragment thus obtained as an amplification template and oligonucleotides
of SEQ ID Nos. 28 and 12 and SEQ ID Nos. 29 and 12 as primer sets was
separated by 1.5% agarose gel electrophoresis, and a 2.5 kb DNA fragment
having a TRP1 gene connected to the human-derived LDH gene and a 2.5 kb
DNA fragment having the TRP1 gene connected to the Leuconostoc
mesenteroides-derived ldh gene were produced by a common method. A
budding yeast strain NBRC10505 was transformed with the 2.5 kb DNA
fragment by a common method, to be tryptophan-nonrequiring.

[0242]The fact that the transformant thus obtained is a yeast having the
human-derived ldh gene or the Leuconostoc mesenteroides-derived ldh gene
introduced at a site downstream of the promoter of PDC 1 gene on
chromosome was confirmed by a method similar to the method of Reference
Example 1.

[0243]Then, by a method similar to that in Reference Example 1, a yeast
strain having the human- or Leuconostoc mesenteroides-derived ldh gene
introduced at a site downstream of the PDC1 gene promoter on chromosome
and having a temperature-sensitive mutation in the pdc5 gene were
prepared. They will be referred to respectively as strains SU019 and
SU024.

[0244]Introduction into the SED1, CWP2 and ENO1 gene locuses was performed
by a method similar to that described in Example 2, except that the
primers were altered. The primers altered will be described below.

Primer for Introduction into SED1 Gene Locus

[0245]PCR was conducted, as the primer shown by SEQ ID No. 30 was used,
replacing the primer shown by SEQ ID No. 14 used in Example 2 in the case
of human-derived ldh gene, and the primer shown by SEQ ID No. 33 was used
in the case of the Leuconostoc mesenteroides-derived ldh gene, and the
strains SU019 and SU024 were transformed, by using the PCR fragment
obtained, respectively to be histidine-nonrequiring. The transformants
obtained will be referred to respectively as strain SU020 (containing
human-derived ldh gene introduced) and strain SU025 (containing
Leuconostoc mesenteroides-derived ldh gene introduced).

Primer for Introduction into CWP2 Gene Locus

[0246]PCR was conducted, as the primer shown by SEQ ID No. 31 was used,
replacing the primer shown by SEQ ID No. 18 used in Example 2 in the case
of human-derived ldh gene and the primer shown by SEQ ID No. 34 was used
in the case of the Leuconostoc mesenteroides-derived ldh gene, and the
strains SU019 and SU024 were transformed, by using the PCR fragment
obtained, respectively to be histidine-nonrequiring. The transformants
obtained will be referred to respectively as strain SU021 (containing
human-derived ldh gene introduced) and strain SU026 (containing
Leuconostoc mesenteroides-derived ldh gene introduced).

Primer for Introduction into ENO1 Gene Locus

[0247]PCR was conducted, as the primer shown by SEQ ID No. 32 was used,
replacing the primer shown by SEQ ID No. 22 used in Example 2 in the case
of human-derived ldh gene and the primer shown by SEQ ID No. 35 was used
in the case of the Leuconostoc mesenteroides-derived ldh gene, and the
strains SU019 and SU024 were transformed, by using the PCR fragment
obtained, respectively to be uracil-nonrequiring. The transformants
obtained will be referred to respectively as strain SU022 (containing
human-derived ldh gene introduced) and strain SU027 (containing
Leuconostoc mesenteroides-derived ldh gene introduced). In addition, the
strains SU020 and SU025 were transformed similarly to be
uracil-nonrequiring, to give respectively strain SU023 (containing
human-derived ldh gene introduced) and strain SU028 (containing
Leuconostoc mesenteroides-derived ldh gene introduced).

Example 4

Lactic Acid Fermentation Test by Batch Culture

[0248]Lactic acid fermentation tests were carried out by batch culture, by
using the strain obtained in Reference Example 1 (SU014) and strains
SU015 to SU028 obtained in Examples 2 and 3. The lactic acid fermentation
medium shown in Table 1 was placed in a 10 mL test tube, and each of the
strains SU014 to SU028 in small amount was inoculated there, and the
mixture was cultured overnight at 30° C. (prepreculture). Then,
100 mL of the fresh lactic acid fermentation medium shown in Table 1 was
placed in a 500-ml Erlenmeyer flask, and each prepreculture solution was
seeded there in the entire amount, and the mixture was cultured under
agitation at 30° C. for 24 hours (preculture). Subsequently, each
of the preculture solutions after preculture for 24 hours was added in
the entire amount into a mini-jar fermenter (manufactured by Marubishi
Bioengineering, volume: 5 L) containing 1 L of the lactic acid
fermentation medium shown in Table 1, and the mixture was cultured at
consistent agitation velocity (120 rpm), ventilation rate (0.1 L/min),
temperature (30° C.) and pH (pH 5) (main culture). The solution
was neutralized with 1N NaOH, and the feed rate was monitored by
measuring weight change with a balance. The culture solution after main
culture for 40 hours was centrifuged, and the supernatant obtained was
filtered through membrane, the accumulated lactic acid concentration was
calculated by the method described in Reference Example 1. The glucose
concentration was determined by using "Glucose Test Wako C" (registered
trade name, manufactured by Wako Pure Chemical Industries).

[0249]The lactic acid/sugar yields calculated from the measurement result
are shown in Table 3. The optical purity of lactic acid was determined by
the method described in Reference Example 1, showing that only L-lactic
acid is detected and D-lactic acid was contained in an amount of less
than detection limit in the case of strains SU014 to SU023, while only
D-lactic acid was detected and L-lactic acid was contained in an amount
of less than detection limit in the case of strains SU024 to SU028.

[0250]The results confirmed that, in continuous culture with simultaneous
filtration of a yeast strain having a lactic acid-producing ability,
lactic acid is obtained at a yield higher by the strains SU015 to SU018
(Xenopus laevis-derived ldh gene-introduced strains), the strains SU020
to 024 (human-derived ldh gene-introduced strains) and the strains SU025
to 028 (Leuconostoc mesenteroides-derived ldh gene-introduced strains),
i.e., yeasts having an introduced lactate dehydrogenase-expressing
cassette having a ldh gene at a site downstream of the promoter of a gene
having an expression amount larger by 10 times or more than the average
relative expression amount of all genes after 50 hours from start of
culture, than by the strains SU014, SU019 and SU024.

Example 5

Lactic Acid Fermentation Test by Continuous Culture

[0251]Filtration continuous culture was carried out in the continuous
culture apparatus shown in FIG. 1, by using the transformant yeast
strains SU015 to SU028 obtained in Examples 2 and 3. The medium used was
a lactic acid fermentation medium in the composition shown in Table 1, of
which the glucose concentration was changed to 70 g/L. The medium was
sterilized by high-pressure steam (2 atm.) at a temperature of
121° C. for 15 minutes before use. Stainless steel and a
polysulfone resin molding were used for the separation membrane element
members. The separation membrane used was the porous membrane prepared in
Reference Example 2. The operational condition in the present Example is
as follows, unless specified otherwise: [0252]Volume of reaction tank:
2 (L) [0253]Volume of fermentation reaction tank: 1.5 (L)
[0254]Separation membrane used: PVDF filtration membrane (prepared in
Reference Example 2) [0255]Effective filtration area of membrane
separation element: 120 cm2[0256]Temperature adjusted to: 30
(° C.) [0257]Ventilation rate of fermentation reaction tank: air:
0.01 (L/min), nitrogen gas: 0.19 (L/min) [0258]Agitation velocity of
fermentation reaction tank: 800 (rpm) [0259]pH adjustment: adjusted to pH
5 with 5N calcium hydroxide [0260]Antifoam: an sterilized antifoam PE-L
(manufactured by Wako Pure Chemical Industries) was added in an amount of
200 μL every 3 hours

[0261]Sterilization: the culture tank including separation membrane
elements and the medium used were all sterilized under high-pressure stem
in an autoclave at 121° C. for 20 min.

[0262]First, each of the strains SU015 to SU028 was cultured overnight in
a test tube containing 10 ml of the lactic acid fermentation medium shown
in Table 1 (preprepreculture). The culture solution obtained was seeded
in the entire amount to 100 ml of the fresh lactic acid fermentation
medium shown in Table 1 and the mixture was cultured under agitation in a
500-ml Sakaguchi flask for 24 hours at 30° C. (prepreculture). The
prepreculture solution was added to 1.5 L of the lactic acid fermentation
medium in the continuous culture apparatus shown in FIG. 1, and the
mixture was cultured for 24 hours, while the reaction tank 1 was agitated
by an attached stirrer 5 at 400 rpm and the ventilation rate, temperature
and pH of the reaction tank 1 were adjusted (preculture). Immediately
after the preculture, the lactic acid fermentation medium was supplied
continuously thereto, and lactic acid was produced by continuous culture,
while the membrane permeation amount was controlled to make the volume of
the fermentation solution in the continuous culture apparatus kept
constant at 1.5 L. The concentrations of lactic acid produced and
residual glucose in the membrane-permeated fermentation solution were
monitored as needed. The accumulated lactic acid concentration was
determined by the method described in Reference Example 1 and the glucose
concentration was determined by using "Glucose Test Wako C" (registered
trade name, manufactured by Wako Pure Chemical Industries).

[0263]The lactic acid/sugar yield and the lactic acid production rate, as
calculated from lactic acid and the glucose supplied calculated from the
glucose concentration, are summarized in Tables 4 and 5, together with
the results obtained in Reference Example 1. Calculation was made
according to Formula 3, and the time in the Table is the time elapsed
after start of culture.

[0264]The results showed that, in continuous culture with simultaneous
filtration of a yeast strain having a lactic acid-producing ability,
lactic acid can be produced at high yield and high production rate
consistently for up to 300 hours by the strains SU015 to SU018, strains
SU020 to SU023 and strains SU025 to SU028, i.e., yeasts having an
introduced lactate dehydrogenase-expressing cassette having a ldh gene at
a site downstream of the promoter of a gene having an expression amount
larger by 10 times or more than the average relative expression amount of
all genes after 50 hours from start of culture. On the other hand, the
yield and the lactic acid production rate declined rapidly after 200
hours, when the strain of Reference Example 1 (SU014) or a strain SU019
or SU024 was used. In addition, measurement of the optical purity of each
sample by the method described in Reference Example 1 showed that only
L-lactic acid is detected and D-lactic acid is present in an amount less
than detection limit in the case of strains SU014 to SU023, while only
D-lactic acid is detected and L-lactic acid is present in an amount less
than detection limit in the case of strains SU024 to SU028.